Application of mass spectrometry in endopolygalacturonases glycosylation site mapping and carbohydrate structure elucidation
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Bioanalytical mass spectrometry is a rapidly developing field due to its wide range of applications. Especially MALDI-MS and ESI-MS have been the essential analytical tools in the areas of proteomics and glycomics. In order to understand the protein functionalities, the extensive post-translational modification events such as glycosylation have to be characterized. The complete characterization of glycosylation is composed of intact protein analysis, glycosylation site mapping and the carbohydrate structure/composition analysis. In each and every step of analysis, mass spectrometry methods can provide detailed information needed. The focus of the research presented in this dissertation is the utilization of sensitive mass spectrometric techniques, mainly MALDI and ESI MS, to characterize the protein glycosylation, the most complex post-translational modification of proteins. The characterization of Botrytis cinerea endopolygalacturonases, BcPG3 and BcPG6, and Aspergillus niger endopolygalacturonase PGC were described. The glycoproteins are first analyzed by MALDI to obtain their molecular masses, followed by trypsin digestion and the resulting peptides are analyzed by LC-ESI MS. The location of glycosylation is identified by using a stepped orifice voltage technique and the structures of the carbohydrates were obtained from ESI MS/MS data. For BcPG3 and BCPG6, multiple N-linked glycosylation sites are identified which are occupied by high mannose N-linked glycans, while one N-linked glycosylation site is identified from A. niger PGC and it is also occupied by high mannose N-linked glycan. In addition, O-linked glycosylations are identified from both BcPG3 and PGC using a convention ESI MS based on the heterogeneity of glycoforms. The location of O-linked glycosylation sites on PGC is further identified from electron-capture dissociation (ECD) MS/MS, which confirms that a single mannose is attached to the residue Thr5.